Long Non coding RNA SNHG16 Functions as Tumor Activator by Sponging hsa-miR-373-3p to Regulate TGFBR2/SMAD Pathway in Prostate Cancer

Background: Long noncoding RNAs (lncRNAs) are one of the major causes of tumorigenesis. However, the roles and mechan isms of lncRNA SNHG16 in prostate cancer (PCa) remain unknown. The purpose of this study was to elucidate the mech anisms of lncRNA SNHG16 in the proliferation and metastasis of human PCa cells. Material and Methods: First, the quantitative polymerase chain reaction (qPCR) was used to measure SNHG16 expression in PCa tissues and adjacent normal tissues (n=80). Down-regulate and over-express SNHG16 in human PCa DU-145 cell. Then cell proliferation was detected by CCK8 assay, cell apoptosis was analyzed by ow cytometry, cell migration were determined by wound healing, and cell invasion was examined by transwell. Western blot assays were used to examine the expression of the TGFBR2, c-MYC, E2F4, SMAD2, p-SMAD2, SMAD3, and p-SMAD3. Second, the targeting relationship between SNHG16 and hsa-miR-373-3p was veried by dual-luciferase reporter assay and rescue experiments. Third, the targeting relationship between hsa-miR-373-3p and TGFBR2 was veried by dual-luciferase reporter assay and rescue experiments. Results: The expression of SNHG16 was signicant increase in PCa tissues (Z=-8.405, P<0.001), and with signicant correlation with patient's age (<60 and ≥ 60 years old, P=0.007). Silencing SNHG16 inhibited DU-145 cell proliferation, migration, and invasion, while induced cell apoptosis signicantly (P<0.01, respectively). Overexpressing SNHG16 promoted cell proliferation, migration and invasion, and reduced cell apoptosis rate (P<0.05, respectively). SNHG16 overexpression observably increased TGFBR2, c-MYC, E2F4, p-SMAD2, and p-SMAD3 expression


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Background Incident cases of prostate cancer (PCa) increased 3.7-fold from 1990 to 2015 [1]. The incidence and burden of PCa are steadily increasing globally. Although there were many treatments for prostate cancer patients, such as such as surgery, radiotherapy, androgen deprivation, chemotherapy, bone-targeting agent etc., it remains the third-leading cause of cancer death in men [2]. Because with the development of genome changes and disease severity, molecular complexity was increased, which affect PCa metastasis, clinical relevance and precision therapy [3]. Hence, carcinogenesis and intrinsic mechanism associated with the tumorigenesis of PCa should be further explored to provide a promising method for detection and intervention.
Studies have proved that non-coding RNA plays a role in the occurrence, progression and treatment of PCa. For example, long noncoding RNAs (lncRNAs) with > 200 bases in length and transcribed from the genomic intergenic regions, which regulated the expression of genes at epigenetic, transcriptional and post-transcriptional levels. LINC00844 regulated global androgen receptor-regulated genes in PCa by facilitating androgen receptor binding to chromatin, and inhibited the progression and metastasis of PCa by activating the expression of the important cancer metastasis suppressor NDRG1 [4]. LncRNA HOXD-AS1 recruited WDR5 to directly regulate the expression of target genes by mediating histone H3 lysine 4 tri-methylation, thus promoting PCa proliferation, castration resistance, and chemo-resistance [5]. LncRNA PCA3 promoted PCa cell growth by down-regulated the tumor suppressor gene PRUNE2 [6]. LncRNA can act as a tumor activator or inhibitor in PCa.
Importantly, LncRNA acts as a competitive endogenous RNA (ceRNA) to regulate MicroRNAs (miRNAs) expression is one of the important mechanisms of its post-transcriptional regulation. MiRNAs are a type of small non-coding RNA with length of 17-22 nucleotides. The production of miRNAs is based on the action of Dicer, which is a kind of RNase that processes the hairpin structured precursors into mature miRNA. Then, the transcribed miRNA suppresses gene expression by recognizing the complementary target site in the 3'untranslated region (UTR) of the target mRNAs [7]. MiRNAs can serve as oncomiRs by targeting tumor suppressor genes and as tumor suppressor by targeting oncogenes, and miRNA-targeted therapeutics have development [8]. LncRNA function as ceRNA by competitively occupying the shared binding sequence of miRNAs, thereby isolating miRNAs and changing the expression of their downstream target genes. Based on the relationship between lncRNAs and miRNAs and mRNAs, He JH et al. screened out 19,075 regulatory relationships which might be involved in the pathogenesis of PCa [9]. The function abnormalities of lncRNA-miRNA-mRNA regulatory network are closely related to the occurrence and development of PCa.
This study intends to analyze the relationship between SNHG16, hsa-miR-373-3p and TGFBR2 to explore its potential mechanism of effect on PCa.

Clinical Tissues Collection
Eighty PCa patients at the Mindong Hospital A liated to Fujian Medical University, between Feb 2019 and Jan 2020, were enrolled into the study. The PCa tumor and adjacent normal tissues samples were collected. All patients were con rmed as PCa by histopathological analysis and without preoperative radiotherapy or chemotherapy. All fresh specimens were stored in liquid nitrogen immediately at −80 °C until use. The detailed criteria for the patients were that the pathological type is adenocarcinoma. The clinical parameters of the enrolled patients such as age, TNM stage and Gleason score et al were recorded and analyzed to correlate the in vitro ndings with the clinical presentations ( RNA extraction and qRT-PCR Total RNA was extracted from PCa tissue and cells by NucleoZOL ® (Gene Co., Ltd., Shanghai, China). Primers were resuspended by adding 250 µL of RNase-free water. Master mix was prepared for each mRNA-speci c assay. Each single reaction included 10 µL of qPCR SYBR® Green Master Mix Universal (Takara Bio, Inc., Japan). About 10 µL of the primer was set for an individual miRNA. RNase free water (3 µL) and RT product (2 µL) were added to a single real-time PCR reaction tube as template.
Reverse transcription (RT) was performed using the RT System Kit (Takara Bio Inc., Tokyo, Japan). The synthesized cDNA was ampli ed by quantitative PCR by using HEAL FORCE (Xianggang, China). The reaction conditions included 42 °C for 60 min, followed by cooling to 4 °C. The resultant cDNA was used as template for subsequent PCR. Forty PCR ampli cation cycles were performed with initial incubation at 95 °C for 10 min and nal extension at 72 °C for 5 min. Each cycle comprised denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The GAPDH mRNA was a control. The relative expression of candidate genes was calculated using the formula ΔΔCq = (C q, target -C q, reference ) PCa -(C q, target -C q, reference ) NC , and the estimated expression ratio was equal to 2 -ΔΔCq [16]. The estimated expression ratio was equal to 2 −ΔΔCq . The primer set of candidate gens: LncRNA SNHG16 FORWARD primer was GTGCCTCAGGAAGTCTTGCC; REVERSE primer was ATCCAAACAAGTTATCAGCAGCAGCAC. TGFBR2 FORWARD primer was GTAGCTCTGATGAGTGCAATGAC; REVERSE primer was CAGATATGGCAACTCCCA GTG. GAPDH FORWARD primer was ATGGGGAAGTGAAGGTCG; REVERSE primer was TTACTCCTTGGAGCCATGTG.
Taqman RT-PCR The RNA harvested from PCa tissue and cells was extracted by the miRNeasy mini kit (Qiagen, USA).
Before RNA extraction, each sample was added with 100 ng exogenous and synthetic Caenorhabditis elegans miR-39 to normalize. Speci cally, sample suspension (50 mg tissue) was mixed with Qiazol lysis reagent (1 mL), incubated or homogenate for 5 min in room temperature, and then mixed with 200 µL cholorform for 3 min. The RNA was separated at 12,000×g for 15 min at 4℃ and collected the aqueous phase. After centrifugal, the supernatant mixed with 100 % ethanol (600 µL). In order to purify RNA, 700 µL supernatant was added to miRNeasy mini spin column and centrifuged at 8,000 × g for 15s at room temperature. Then, miRNA was eluted by the addition of 30 µl RNase-free water. Concentration of RNA was measured by nanodrop.

Cell culture and transfection
Human PCa DU-145 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI-1640 medium (Gibco, USA) and cultured in the media with 10% FBS (PAN biotech, Germany), 4 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a humidi ed atmosphere of 5% CO 2 .

CCK-8 assay for cell proliferation
The transfection cells were determined by Cell Counting Kit-8 assay (Dojindo, Japan). DU-145 cell suspensions (1×10 4 /mL) were transferred to 96-well plates and incubated for 24, 48, 72, and 96 h. Each well was added with 10 μL of CCK-8 and incubated for 4 h. The values of each well were measured by a microplate reader (PERLONG, Beijing, Chia) at 450 nm. All experiments were repeated three times.

Flow cytometric analysis for cell apoptosis
The 1×10 5 cells/ well were cultured in 24-well plates and transfected until the con uence reached 60%-70%. After transfecting with plasmid for 48 h, the cells were washed two times with PBS and centrifuged for 5 min at 1000 rpm. According to the instruction, the cells were then incubated with 5 μL of Annexin V-FITC for 15 min and 10 μL of PI for 5min at room temperature and dark (BD Biosciences, USA). Finally, the cells were detected by ow cytometry (Becton Dickinson, USA). All experiments were repeated three times.
Wound healing assay for cell migration Wound healing test was used to measure cell migration. After transfecting with plasmid for 48 h cells were reaped. A total of 3×10 5 cell/well were inoculated into 24-well plates, which each well has cell plugin. Wound healing experiment was carried out when all the cells were adherent and just full. After incubation for 24 h, the cells were counted under the microscope. All experiments were repeated three times.
Transwell assay for cell invasion Cell invasion was assayed using a Transwell chamber (8 μm pore size, Corning) deposited with Matrigel (BD Biosciences, USA). After transfecting with plasmid for 48 h, 1×10 5 cell/well was inoculated into Transwell's upper chamber. The lower chamber was added with complete media. After 24 h, cells that remained in the upper membrane were removed. The migrated cells were incubated with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 15 min, and counted in the microscope. All experiments were repeated three times.

Western blot
After transfecting 48 h, DU-145 cells were collected. RIPA buffer (Takara Bio Inc., Tokyo Japan) and protease inhibitor (Roche Diagnostics) were used for cell lysis. BCA assay kit was used to detect protein concentration (Epizyme, China). The protein samples were separated using SDS-PAGE and shifted to PVDF. The PVDF membranes were saturated with blocking buffer (5% skim milk in 20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature and incubated with primary antibodies (1:1000) at 4 °C overnight. The antibodies of TGFBR2, c-MYC, E2F4, SMAD2, p-SMAD2, SMAD3, p-SMAD3, and βactin (Cell Signaling Technology, Beverly, MA, USA) were used. After washing two times with PBS, the cells were incubated with secondary-HRP-antibodies (Protein-Tech, USA) for 2 h at room temperature. The membranes were added with ECL Western blotting reagents (GE Healthcare, USA) to detect protein levels. The bands were imaged by Tanon 5200 Biotanon. Image J software was used to calculate the gray value.

Statistical analysis
All experiments were analyzed by the IBM SPSS Statistics 20.0 software (IBM Corp., Armonk, New York, USA). If data followed normal distribution, the one-way ANOVA analysis with LSD post-hoc test was performed. If the data did not follow normal distribution, Mann-Whitney U test was used. Spearman correlation was used to analyze the correlation between SNHG16 and hsa-mir-373-3p, hsa-mir-373-3p and TGFBR2. Chi-square test was used to descriptive analysis. Datas were presented as mean ± standard deviation, and P < 0.05 indicated signi cant difference.

Results
The correlation with patient's characteristics and SNHG16, hsa-miR-373-3p and TGFBR2 expression Division of the 80 patients into those with high expression of SNHG16, hsa-miR-373-3p and TGFBR2, and those with low expression was done using the difference value to assess the link between the molecule and the clinical characteristics (Table 2). First, we analyzed the expression of SNHG16, hsa-miR-373-3p and TGFBR2 in PCa according to the patient's age (age groups: <60, 60≥years old). SNHG16 expression was higher in patient's age ≥60 years old (P=0.007), whereas in these two groups of patients (<60 and 60≥ years old) hsa-miR-373-3p and TGFBR2 expression lost its signi cance (P=0.789, P=0.606, respectively). Second, the expression of SNHG16, hsa-miR-373-3p and TGFBR2 were analyzed according to the Gleason score (Gleason score: <7, >7). The up-regulation of SNHG16 and TGFBR2 in PCa patients were no statistical correlation with Gleason score (All P > 0.05). However, the down-regulation of hsa-miR-373-3p were closely linked to Gleason score (P = 0.024). Third, SNHG16, hsa-miR-373-3p and TGFBR2 expression were analyzed according to the TNM stage (TNM stage: T1, T2, T3/4). The results showed that there were no signi cant correlation with gene expression and TNM stage (All P > 0.05).
The high expression of SNHG16 in PCa tissues promoted tumor cell proliferation, migration and invasion by regulating TGFBR2/SMAD signaling The result of qRT-PCR showed SNHG16 was signi cantly increased in PCa tissues comparison with adjacent normal tissues (P<0.001) (Fig. 1A). SNHG16 might play an important role in tumor progression [10]. In order to study the role of SNHG16 in PCa, we knocked down and overexpressed SNHG16 in DU-145 cells (Fig. 1B). Then, we further explored the biological role of SNHG16 in DU-145 cell. SNHG16 knockdown signi cantly suppressed cell proliferation (P<0.001), and SNHG16 overexpression signi cantly promoted cell proliferation (P<0.001) (Fig. 1C). Knockdown of SNHG16 signi cantly increased cell apoptosis (P<0.001), and the overexpression of SNHG16 signi cantly inhibited cell apoptosis (P<0.01) (Fig. 1D). The transwell test revealed that inhibition of SNHG16 expression signi cantly reduced cell invasion (P<0.01), and up-regulation of SNHG16 expression notably increased the number of cell invasion (P<0.05) (Fig. 1E). The wound healing test showed SNHG16 inhibition signi cantly decreased cell migration (P<0.001), and SNHG16 up-regulation markedly increased the number of cell migration (P<0.001) (Fig. 1F).
Next, the protein expression levels of c-MYC, TGFBR2, E2F4, SMAD2, p-SMAD2, SMAD3, and p-SMAD3 were determined by Western blot. The protein expression levels of c-MYC, TGFBR2, p-SMAD2, p-SMAD3, and E2F4 signi cantly decreased in the sh-SNHG16 group compared with those in the sh-NC group (P<0.001), but these proteins were signi cantly increased in the OE-SNHG16 group compared with those in the OE-NC group (P<0.001) (Fig. 1G). However, the SMAD2 and SMAD3 expression were not affected by SNHG16 (Fig. 1G). Our results indicated that the part effect of SNHG16 promoted cell proliferation, migration and invasion via activating the TGFBR2/SMAD signaling pathway.

Discussion
PCa is one of the main lethal diseases among the cancer mortality rate of men, and the incidence rate increased signi cantly than in the past [1,2]. Therefore, a novel target should be identi ed to improve the prognostic outcome of patients with PCa for developing effective therapeutic treatments. The functions of lncRNAs in PCa tumorigenesis have received increasing attention. LncRNAs can fold into secondary and tertiary structures and function as modulators of biological processes in most cancer types, including PCa [4][5][6]. Several lncRNAs, such as LINC00844 [4], HOXD-AS1 [5], PCA3 [6] and SChLAP1 [17] etc., are dysregulation in the tumorigenesis of PCa and play oncogenic or tumor-suppressive roles. Here, a kind of lncRNA SNHG16 was found as a potential oncogene modulates PCa cell biological function via hsa-miR-373-3p /TGFBR2/SMAD signaling axis.
A novel lncRNA SNHG16 of PCa has been originally explored as a tumor activator in neuroblastoma and is located on chromosome 17q25.1. SNHG16 was abnormally expressed in patients with aggressive neuroblastoma and acted like an oncogene [18]. SNHG16 might be a prognostic indicator as a potential tumor target and functional regulator in tumorigenesis. The upregulated expression of SNHG16 signi cantly associated with invasion depth, lymph node metastasis, TNM stage and histological differentiation of gastric cancer [19]. However, SNHG16 was markedly down-regulated and induced tumor-suppressing effects in hepatocellular carcinoma [20]. SNHG16 in multiple cancer types can be either positively or negatively regulated possibly due to the intrinsic subtypes of speci c cancer. In this study, the up-regulation of SNHG16 expression in PCa tissues was signi cantly related to the patient's age (< 60 and 60 ≥ years old, P = 0.007), and the higher the high expression rate of SNHG16 when the Gleason score > 7 or T3/T4 stage. Older age was associated with the Gleason score ≥ 7 or prostatectomy T3/T4 [21]. Age was an independent predictor of shorter PCa-speci c survival patients diagnosed with de novo metastatic PCa [22]. Besides, the overexpresion of SNHG16 would induce PCa cell proliferation, migration and invasion. In consequence, SNHG16 might play the oncogenic effects in PCa development or progression.
Recent research reports, SNHG16 over-expressed in PCa patients and play a role in promoting cell proliferation by regulating GLUT-1 expression and glucose uptake [10]. Here, we found SNHG16-incuded DU-145 cell proliferation, migration and invasion via up-regulating c-MYC, TGFBR2, E2F4, p-SMAD2, and p-SMAD3 expression. The c-MYC is a proto-oncogene and plays a role in cell cycle progression, apoptosis and cellular transformation. The high expression of c-MYC could promote the development of PCa by the transcription of the androgen receptor gene and enhance the stability of the full-length androgen receptor and androgen receptor splice variants proteins [23]. E2F4 were overexpressed in PCa epithelial cells [24], and regulated cell cycle by forming complexes with P130 [25]. E2F5 overexpression induced uncontrolled cellular proliferation by up-regulating phosphorylation of SMAD3 and p38 in PCa [26]. The endogenous SMAD2/3 interacted with PKCε to cause SMAD3 to bind to the promoter of glycolysis genes, induced the expression of glycolysis genes HIF-1α, HKII, PFKP and MCT4 and promoted aerobic glycolysis, thereby promoting PCa cell proliferation [27]. In addition, overexpression of SMAD3 could also enhance aerobic glycolysis and PCa cell proliferation in an independent manner that activated protein kinase D or TGF-β [27]. However, SMAD3 or TGFBR2 null were signi cantly reduced the mass and microvascular density of PCa xenograft tumors [28]. Therefore, SNHG16 increased the expression of c-MYC, TGFBR2, E2F4, p-SMAD2, and p-SMAD3, which was an important mechanism for its carcinogenic effects.
Numerous lines of evidence showed that lncRNAs can function as ceRNA by sequestering miRNAs [29].
MiRNAs are single-stranded, noncoding RNAs that regulate gene expression via the mechanisms of conserved across metazoans. In this study, hsa-miR-373-3p was signi cantly decreased in PCa tissues, and had signi cantly negative correlations with SNHG16 (r = -0.544). In addition, the low expression of hsa-miR-373-3p was signi cantly related to the Gleason score, and the low expression rate of hsa-miR-373-3p when the T2 or T3/T4 stage increased. Hsa-miR-373-3p might act as a tumor suppressor in PCa.
However, in testicular germ cell tumors, miR-373 could promote proliferation and tumorigenesis of testicular germ cell by neutralizing p53-mediated CDK inhibition [32]. Hence, hsa-miR-373-3p has a dual function as a promoter or suppressor in different cancers. TGFBR2 was an important cancer driver in gastric cancer and liver tumorigenesis [33,34]. Besides, the down-regulation of TGFBR2 inhibited the expression of SMAD-dependent metastasis-promoting genes PTHrP, PAI-1 and ANGPTL4, and breast cancer cell invasion [30]. The effects of TGF-β/TGFBR2 signaling in PCa progress was strongly dependent on the stage of development, which acted as a tumor suppressor in early stages and acted as a promoter in later stages [35]. In this study, the high expression of TGFBR2 might be related to the inclusion of more advanced patients.
In conclusion, this study revealed that the high expression of SNHG16 promoted the proliferation, migration and invasion of turomr cell via functionally sponging hsa-miR-373-3p and regulating the TGFBR2/SMAD pathway, especially in patients with advanced PCa. However, this study has several limitations. First, the study only analyzed the association of SNHG16, has-miR-373-3p and TGFBR2 in DU-145 cell line. More than one cell lines should be studied. Second, we did not design the animal model to verify the role of SNHG16. In the future, additional clinical and animal experiments are needed to demonstrate that SNHG16 act as a ceRNA and regulates the hsa-miR-373-3p /TGFBR2 axis, thereby affecting the biological function of PCa cells.

Conclusions
LncRNA SNHG16 acted as a ceRNA to enhance the proliferation, migration, and invasion of PCa cells by modulating the hsa-miR-373-3p/TGFBR2 axis. These data provide novel insights into SNHG16, which may be a novel target for treatment of PCa. Availability of data and materials: All data analyzed and displayed in the present manuscript are available from the corresponding author upon reasonable request.

Declarations
Competing interests: The authors declare that they have no competing interest.